Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase  Rémi Buisson, Jessica L. Boisvert, Cyril H. Benes, Lee Zou  Molecular Cell  Volume 59, Issue 6, Pages 1011-1024 (September 2015) DOI: 10.1016/j.molcel.2015.07.029 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 Acute ATR Inhibition Exerts Two Distinct Effects on S-Phase Cells (A) U2OS cells were cultured in BrdU for 36 hr, treated with DMSO or ATRi 1 (10 μM VE-821), and analyzed for BrdU and γH2AX by immunostaining. (B) Quantification of the BrdU intensity of 1,000 U2OS cells treated with DMSO or ATRi. Black lines indicate median BrdU intensities of BrdU-positive cells in various cell populations. (C) Quantification of the BrdU and γH2AX intensities of 1,200 U2OS cells treated with DMSO or ATRi. Cell subpopulation 1 displayed less ssDNA at 8 hr than at 2 hr. Cell subpopulation 2 displayed very high levels of ssDNA and became γH2AX positive at 8 hr. (D) Levels of RPA32, pRPA32, and γH2AX in the soluble and chromatin fractions of ATRi-treated cells were analyzed by western blot. (E) The levels of chromatin-bound RPA32 and pRPA32 were analyzed in cells treated with ATRi 2 (AZ20) and ATRi 3 (EPT-46464). See also Figure S1. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 ATR Suppresses ssDNA Accumulation in Early S Phase (A and B) Quantification of chromatin-bound RPA, EdU incorporation, and DNA contents of 5,000 U2OS cells treated with DMSO or ATRi 1 (10 μM VE-821). Cells were color coded according to the intensity of RPA staining (left). (C) T98G cells were released synchronously from G0 and analyzed for EdU incorporation at the indicated times. (D) Staining intensity of chromatin-bound RPA was analyzed at different stages of the cell cycle after ATRi or DMSO treatment. Red lines indicate mean RPA intensities in various cell populations. ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S2. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 ATRi Suppresses DNA Damage by Promoting RRM2 Accumulation and Limiting Origin Firing (A) T98G cells were released synchronously from G0 in the presence or absence of ATRi 1 (10 μM VE-821). Levels of RRM2, γH2AX, RPA70, RPA32, RPA14, and Cyclin A were analyzed during the time course. (B) Asynchronously growing U2OS cells were treated with ATRi 1 (10 μM VE-821) or Chk1i 1 (2 μM MK-8776). Levels of RRM2 and E2F1 were analyzed at the indicated times. (C) Levels of RRM2 and E2F1 were analyzed in U2OS cells treated with DMSO or ATRi in the presence of cycloheximide (CHX). Relative levels of RRM2 and E2F1 were quantified from three blots (n = 3). Error bars, SD. (D) U2OS cells transfected with empty vector or E2F1-expressing plasmids were treated with ATRi for 8 hr. Levels of RRM2, E2F1, and γH2AX were analyzed. (E) U2OS cells were treated with the indicated inhibitors for 8 hr. Levels of RRM2, E2F1, and γH2AX were analyzed. (F) U2OS cells were treated with DMSO or ATRi for 8 hr in the presence or absence of MG132 or MLN4924. (G) U2OS cells infected with HA-RRM2-expressing retrovirus or control virus were treated with ATRi for 8 hr. Levels of RRM2 and γH2AX were analyzed at the indicated times. (H) A model in which ATR coordinates RRM2 accumulation and origin firing in early S phase is shown. See also Figure S3. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 ATRi-Treated Cells Recover via a Chk1-Mediated Mechanism (A) U2OS cells were treated with DMSO, ATRi 1 (10 μM VE-821), or Chk1i 1 (2 μM MK-8776). Levels of RPA32, pRPA32, and γH2AX were analyzed at the indicated times. (B) U2OS cells were treated with increasing concentrations of ATRi or Chk1i for 24 hr and then cultured in inhibitor-free media. Cell survival was analyzed 4 days after treatment. Error bar, SD (n = 3). (C) U2OS cells were treated with DMSO, ATRi, or Chk1i for 8 hr. BrdU and γH2AX intensities were quantified in 1,200 cells at the indicated times. (D) U2OS cells were treated with ATRi or Chk1i. Levels of chromatin-bound RPA were analyzed at the indicated times. (E) U2OS cells were treated with ATRi, and levels of pChk1 and CDC25A were analyzed at the indicated times. (F) Levels of CDC25A in U2OS cells treated with ATRi or Chk1i were compared at the indicated times. (G) A model in which Chk1 promotes the recovery of ATRi-treated cells with moderate ssDNA is shown. See also Figure S4. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 Regulation and Function of Chk1 during Recovery (A) Levels of pDNA-PK, pATM, pChk1, and CDC25A were analyzed in ATRi-treated U2OS cells at the indicated times. (B) U2OS cells were treated with ATRi, ATMi, DNA-PKi, or the combinations of these inhibitors. Levels of pDNA-PK, pATM, pChk1, and CDC25A were analyzed 8 hr after treatment. (C) The percentage of replication tracts containing fired origins was determined in RPE1 cells treated with DMSO or ATRi at the indicated times. Error bars, SD (n = 3 experiments). ∗∗p < 0.01; ∗∗∗p < 0.001. (D) RPE1 cells were treated with DMSO or various inhibitors as indicated. The inter-origin distance was analyzed using a DNA fiber assay at the indicated times. Error bars, SEM (n = 25 to 67 as indicated). ∗∗∗∗p < 0.0001; n.s., not significant. (E and F) RPE1 cells were treated with DMSO or various inhibitors as indicated. The length of continuous replication tracts was determined using a DNA fiber assay at the indicated times (>600 forks per condition, n = 3 experiments). See also Figure S5. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 ATRi Selectively Kills Cells under High Replication Stress (A) U2OS cells were treated with ATRi for 2 hr in the presence of increasing concentrations of HU. The BrdU intensity of 1,000 U2OS cells was quantified. Black lines indicate mean BrdU intensities of BrdU-positive cells in various populations. (B) U2OS cells were treated with ATRi or Chk1i for 16 hr in the presence of increasing concentrations of HU. Cell death was measured by the TUNEL assay. Error bars, SD (n = 3). (C and D) U2OS cells were induced to overexpress Cyclin E or left uninduced, and treated with ATRi or Chk1i for 16 hr. Levels of Cyclin E and γH2AX were analyzed by western blot (C). Fractions of TUNEL-positive cells were quantified (D). Error bars, SD (n = 3). (E and F) T98G, RPE1, and MCF10A cells were treated with DMSO or ATRi. Levels of ssDNA were analyzed by native BrdU staining 2 hr after ATRi treatment (E). Yellow lines indicate mean BrdU intensities of BrdU-positive cells in various populations. Levels of cell death were measured by TUNEL assay at the indicated times (F). Error bars, SD (n = 3). (G) Quantification of ssDNA, γH2AX, and cell survival of ten colorectal cell lines after ATRi treatment. Levels of ssDNA and γH2AX were analyzed 2 and 16 hr after the indicated treatments, respectively. Fractions of ATRi-treated cells displaying stronger BrdU staining than untreated cells were determined (see Supplemental Experimental Procedures). Cell survival was analyzed 6 days after the indicated treatments using CellTiter-Glo. See also Figure S6. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 Modeling the Roles for ATR, DNA-PK, and Chk1 in Countering Replication Stress (A) A fraction of early S-phase cells are particularly vulnerable to ATR inactivation. (B) ATRi selectively kills cells under high replication stress, whereas Chk1i induces cell death even in cells in which replication stress is moderate. (C) ATRi-induced ssDNA is an indicator of replication stress that may predict the ATRi sensitivity of cancer cells. Molecular Cell 2015 59, 1011-1024DOI: (10.1016/j.molcel.2015.07.029) Copyright © 2015 Elsevier Inc. Terms and Conditions